Nuclear magnetic resonance (NMR) spectroscopy utilizes the magnetic properties of nuclei to provide information on the chemical characteristics of a molecule. When placed in a magnetic field, NMR active nuclei absorb and emit energy at a frequency characteristic of the isotope. Typically, a target is placed in a strong magnetic field that causes the generally disordered and randomly oriented nuclear spins of the atoms to become aligned with the applied magnetic field. One or more radio frequency (RF) pulses are transmitted into the target, perturbing the nuclear spins. As the nuclear spins relax to their aligned state, the nuclei emit RF energy that is detected by receiving coils arranged about the target. The energy absorption and the intensity of the resulting signal are proportional to the strength of the magnetic field.
Depending on the local chemical environment, different protons in a molecule resonate at slightly different frequencies. Because the frequency is proportional to the strength of the magnetic field, it can be converted into a field-independent dimensionless value known as a chemical shift. The chemical shift may be reported as a relative measure from a reference resonance frequency. For example, the chemical shift may be determined as a difference between the frequency of the signal and the frequency of the reference divided by the frequency of the reference signal. Commonly measured reference nuclei are hydrogen-1 (1H) and carbon-13 (13C), though nuclei from isotopes of many other elements can also be used as references. The frequency shifts generally are extremely small (typically hundreds of Hertz) in comparison to the frequency of the reference signal (typically hundreds of megahertz), and thus, are generally expressed as parts per million (ppm).
Analysis of a one-dimensional (1D) NMR spectrum provides information related to the number and the type of chemical entities in a molecule. For example, with reference to FIG. 1, a 1D NMR spectrum is shown of an equimolar mixture of twenty-six small molecule standards using a 1H reference nuclei. Multi-dimensional NMR involves a series of 1D experiments. Each experiment consists of a sequence of RF pulses with delay periods between each sequence. The use of pulses of different shapes, frequencies, intensities, and durations in specifically-designed patterns or pulse sequences allows a determination of different types of information about the molecule and distinguishes different multi-dimensional NMR types. For example, there are multiple types of two-dimensional (2D) NMR spectroscopy including correlation spectroscopy, J-spectroscopy, exchange spectroscopy, nuclear Overhauser effect spectroscopy, total correlation spectroscopy, heteronuclear correlation experiments, etc. Heteronuclear correlation experiments may include heteronuclear single quantum coherence (HSQC), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), etc. 2D NMR spectrum provide more information about a molecule than 1D NMR spectra. For example, with reference to FIG. 2, a 2D NMR spectra is shown of the equimolar mixture of twenty-six small molecule standards shown in FIG. 1 overlaid onto a spectrum of aqueous whole-plant extract from Arabidopsis thaliana. Peaks from covalently bonded 1H-13C pairs are identified on the basis of simultaneously satisfying positions on the x-axis (frequency of the 1H nucleus) and y-axis (frequency of the 13C nucleus).
1D 1H NMR spectroscopy has been used as an analytical tool for identifying small molecules and measuring their concentrations. Traditionally, quantitative analysis by NMR has been restricted to relatively simple mixtures with minimal peak overlap because overlapped peaks do not scale in a discrete linear fashion that typifies well-isolated peaks. Instead, overlapped peaks scale as the sum of the total overlapped resonance. As a result, multivariate and correlation statistics are reporters of overlapped spectral density rather than the concentrations of specific compounds. Thus, although peak overlap does not interfere with the reproducibility of traditional analyses, it does prevent accurate quantification.
Recently, interest has surged in using NMR for high-throughput analysis of complex biological processes at the metabolic level. These studies, defined as “metabolomics” or “metabonomics”, place an emphasis on biomarker discovery or disease classification and are typically centered on unfractionated biological fluids and tissue extracts. 1D 1H NMR spectra of these types of samples typically contain hundreds of overlapping resonances (see FIG. 1) that make traditional NMR-based analytical practices, such as resonance assignment and accurate peak integration, impossible or impractical.
Using 2D 1H-13C NMR (see FIG. 2), peak overlap is reduced. However, applications of multidimensional NMR in the metabolomics literature have been largely restricted to qualitative analyses generally for two reasons. The first reason is that 2D 1H-13C cross-peak intensities (or volumes) are influenced by a greater number of variables (e.g. uneven excitation, non-uniform relaxation, evolution times, mixing times, etc.) than are 1D 1H NMR peaks. The non-uniform behavior makes it difficult to translate peak intensities into metabolite concentrations. A second reason is that 2D 1H-13C NMR spectra usually require more time to collect than 1D 1H spectra. Long acquisition times are impractical for metabolomics studies that require the analysis of hundreds of samples. Thus, a method and a system to support metabolomics studies using NMR data is needed.